Development of an Enzyme-linked Aptamer-based Assay for the Detection of Tetracycline
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Abstract:
The Mannich method was employed to conjugate the tetracycline (TC) to horseradish peroxidase (HRP). The optimal coating concentration of streptavidin (SA) was 8 μg/mL and aptamer concentration was 20 nmol/L, which were determined by chequerboard titration. The optimized assay conditions were investigated by single-factor experiments. Coating buffer carbonate buffer (CB, 0.05 mol/L, pH 9.6), phosphatic buffer solution (PBS) plus 5 mmol/L MgCl2 and PBS were chosen as coating buffer assay buffer, and standard dilution buffer, respectively.was, The best dilution ratio of TC-HRP and incubation time of aptamer were 1/50 and 1 hour, respectively. An enzyme-linked aptamer assay (ELAA) method was thus established for the determination of TC. The half inhibition concentration (IC50) of the proposed method was 0.705 μg/mL, and the limit of detection (LOD) was 2.5 ng/mL. Compared with other structural analogues (eg. doxycycline, oxytetracycline and chlortetracycline) of TC, there were no conspicuous cross reactions except chlortetracycline (25.9%). This direct competitive ELAA method is specific and highly sensitive that could meet the requirements of quantitative analysis of TC, which is suitable for the rapid detection of TC in variable food samples.
